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Sequence alignment

Pairwise alignment

A) DOT PLOT

The easiest way to align two sequences is to use a dotplot. In its most straight forward implementation the two sequences to be aligned are written along the coordinate axis.

In more realistic implementations a window of 5 to 20 nucleotides or amino acids is slid along one of the axes (i.e., sequences) and compared to every possible window on the other axis (sequence). The dot intensity is adjusted to reflect the percent identity (or similarity) in the two windows.

See the Dotlet exercises below.

Optimal global and local alignments.

There are many different algorithms to calculate pairwise sequence alignments. For two sequences it is "easy" to calculate an optimal global alignment. (According to the motto: "It can be easily shown" -- see here). The so called Needleman-Wunsch algorithm is widely used, it optimizes a positive alignment score, a related (and under some conditions equivalent approach) is to minimize the differences between to sequences.

Multiple Sequence Alignments

CLUSTAL, CLUSTALW and CLUSTALX

Usually global alignments are the easiest to calculate (local see below)

One of the easiest to use, most sophisticated, and most versatile alignment programs is clustalw

(Higgins DG, Sharp PM (1988) CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73:237-244;
Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673-4680).

Clustalw uses a very simple menu driven command-line interface, and you also can run it from the command line only (i.e. it is easy to incorporate into scripts.)

Clustalx uses the same algorithms as clustalw.  However, it has a much nicer interface, it displays information on the level of similarity, and it uses color in the alignment.  Especially for amino acids the use of color greatly enhances the ability to recognize conservative replacements. Clustalx is available for different platforms at the ebi's ftp site (follow your platform, clustalx is stored in the clustalw folders)

Clustal reads and writes most formats used by different programs.  The easiest format is the FASTA format:

> name of sequence or any other information goes in the first line. This line starts with ">". The line can be longer than 80 characters. The first line ends with the first paragraph sign.p
The second line contains the sequence itself; numbers and other non standard characters are ignored. Be careful if you download sequences. Often the transfer programs introduce paragraph signs every 100 characters, and the end of a command line frequently ends up as the beginning of the sequence.
All sequences to be read should be in a single file.

(sample clustalw input file)

(sample clustalw output file)

Clustal also reads aligned sequences.  If you input aligned sequences you can go directly to the tree section.
!! Be careful if you make a mistake, and the sequences are not aligned, your tree will look strange!!
!!! ALWAYS CHECK YOUR ALIGNMENT!!!

Clustal also is useful to reformat and edit alignments, it is very forgiving in reading formats, e.g., you can open the clustal format (*.aln) in a text editor and delete columns and reload the file into clustalw, and output it in the other formats available.

For calculating an alignment, you can select different substitution matrices, and gap penalties (end-gaps can be considered differently!)

Clustal is better than its reputation. It is doing a great job in handling gaps, especially terminal gaps, and it makes good use of different substitution matrices.

To align sequences clustal performs the following steps:

1) Pairwise distance calculation
2) Clustering analysis of the sequences
3) Iterated alignment of two most similar sequences or groups of sequences.

It is important to realize that the second step is the most important. The relationships found here will create a serious bias in the final alignment. The better your guide tree, the better your final alignment. You can load a guide tree into clustal. This tree will then be used instead of the neighbor joining tree calculated by clustalw as a default. (The guide tree needs to be in normal parenthesis notation WITH branch lengths).

Other programs often used for multiple sequence alignment
(We will not use these program in this course; if you are already confused by the information provided, skip to the assignments):

A program available via the www is SAM (sequence alignment and modeling system) by Richard Hughey, Anders Krogh, Christian Barrett, & Leslie Grate at UCSC. The input consists of a multiple sequence file (aligned or not aligned) in FASTA format. The program uses secondary structure predictions, neighboring sites, etc. to place gaps. The program can be accessed using netscape at " http://www.cse.ucsc.edu/research/compbio/sam.html ".

PILEUP in the GCG package generates alignments that are very similar to clustalw. The TREE programs in GCG are currently considered by many to be worthless (UPGMA).  It is planned -since over three years- to incorporate PAUP into GCG in the "near future".

• Advantage: you get to know your sequences and the motives that are conserved are reliably aligned
• Problems: time consuming, Macaw has the tendency to crash.

Alignments by Eye:
On PCs there is a DOS program called the Eyeball Sequence Editor (ESEE) that allows to simulataneously align nucleotid and encoded protein sequences. Needs some getting used to.

One useful sequence editor is seaview, the companion sequence editor to phylo_win. It runs on PC and most unix flavors, and is the easiest way to get alignments into phylo_win.

Assignments:

The Swiss Institute for Bioinformatics provides a JAVA applet that perform interactive dot plots. It is called Dotlet. The main use of dot plots is to detect domains, duplications, insertions, deletions, and, if you work at the DNA level, inversions (excellent illustrations of the use of dot plots are given on the examples page). Currently, the applet will work in Internet Explorer ONLY.

1. Comparing yeast ATPase catalytic subunit with yeast HO endonuclease. Go to the applet and input the sequences: here, here, here and here. Careful, once you leave the webpage, the back arrow will only return you to the applet, but you have to input the sequences again (so make sure that your applet is in a separate browser window). Also, when you input sequences make sure you paste sequence only, without a sequence description line.
   
   
2. Select Neurospora A-subunit and the yeast subunit with intein. Select a window size between 9 and 15 and click "compute". The program will compare every window of the chosen size in one sequence to all the possible windows in the other sequence. On the right you see a histogram that describes how often window pairs with the indicated score occurred. The sliding bars below and above the histogram let you select the colors with which matches are depicted. (I like black for matches, white for mismatches better than the default).
   
   If you click on the dot plot panel, the alignment window at the bottom aligns the two sequences accordingly. You can fine-tune the alignment using the arrows.
   
   Which sequence positions in the yeast sequence represent the intein?
   
   
3. If you compare the HO endonuclease (sex change enzyme) to the intein only, does the complete intein sequence match to something in the HO endonuclease? Is there a part of the sequence in the HO endonuclease that might correspond to an extein?
   
   
4. Comparison of nucleotide sequence with introns vs. protein sequence it codes. (If you are low on time skip 4-12, results are in class12, mcb221, choose a few examples that you find interesting).
   Dot plots have many different applications. One of them is to analyze and visualize the intron exon structure of genes. In dotlet, if you use a nucleotide sequence for the first sequence, and a protein sequence for the second, the program will compare the translation in all three frames to the protein sequence. Load the following two sequences into dotlet:
   
   A) the genomic sequence from Arabidopsis thaliana containing the gene encoding the vacuolar ATPase (sequence below at end of page), the given sequence is the reverse complement of a sequence that is part of chromosome 1.
   
   B) the protein sequence as translated from the cDNA sequence as given in GI 3334404
   
   
5. How many exons are in the gene ? (Is this the same result we got Wednesday afternoon?)
   
   
6. Are neighboring exon sequences always in the same reading frame? (Use the mouse pointer to place the blue cross-hairs on the diagonal and then use the arrow key until one of the three frames matches to the protein sequence.) Try this for a couple of exons. WHY?
   
   
7. Using BLAST to compare nucleotide and protein sequences
   An alternative approach is to use the genome sequence in a blast search of a protein databank. A drawback is that small exons might be below the significance level.
   
   Use the nucleotide sequence given below and compare it to the Swiss Protein database.
   Which BLAST program do you use?
   
   
8. Should you turn off the low-complexity filter?
   
   
9. How many exons can you identify?
   Note that you don't need to have the cDNA from the organism whose gene you are studying in the database. The matches to related organisms (in this case plants) are sufficient to identify the exons.
   
   
10. Repetitive proteins in Dotlet
    Using dotlet load GI 30686594 and GI 19887539 (again omit the labels from the sequence, but give them a name so you can recognize them :)).
    Compare the Arabidopsis proteolipid against itself. Do you see any repetitive units? How many?
    Does the choice of scoring matrix make a difference?
    
    
11. Compare the Methanopyrus sequence against the one from Arabidopsis. How many equivalents to the single repeat unit in Arabidopsis do you find?
    
    
12. How many repeats do you identify when you compare the Methanopyrus sequence against itself?

Task 2)

Start clustalx by double-clicking the icon;
load globin.pep (right click, save to your computer) into the program;
calculate an alignment and save the result under different names in the different possible formats.  Using a text editor (MS Word) and a non-proportional font (i.e., a type of Courier), inspect the different formats.

*.MSF is read by GCG; *.PIR output is like FASTA - format, but the seq. all have equal lengths, you have to merge the first and second line in order to be FASTA compatible (you also could use the GDE format and using a text editor replace % with >).
Phylip (*.phy) is the "new" phylip - interlaced format. 

You can use the input/output options to reformat an alignment. Some programs require specialized formats. You can use a text editor like MSWord to get your alignment into the desired format, but things are certainly much easier, if you start out with a format close to the desired one.

Task 3)

Task 4)

Task 5) (we skip this exercise)

Task 6)

If you have time, explore one of the many different multiple sequence alignment websites available. Use a multiple sequence file of your choice (see above for examples). Possible programs include:

• Muscle http://www.ebi.ac.uk/Tools/muscle/index.html
• TCoffee http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi
• MAFT http://mafft.cbrc.jp/alignment/server/
• Guidance http://guidance.tau.ac.il/ (article at http://nar.oxfordjournals.org/cgi/content/abstract/gkq443v1)
• Head or Tail (same server as above) (article at http://mbe.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=17387100)